TRACELESS REDUCTIVELY CLEAVABLE LINKER MOLECULES FOR PEPTIDE PURIFICATION
20210332082 · 2021-10-28
Assignee
Inventors
Cpc classification
C07K1/22
CHEMISTRY; METALLURGY
C07D233/64
CHEMISTRY; METALLURGY
C07C247/18
CHEMISTRY; METALLURGY
C07D239/26
CHEMISTRY; METALLURGY
C07D237/08
CHEMISTRY; METALLURGY
C07D213/74
CHEMISTRY; METALLURGY
C07C323/63
CHEMISTRY; METALLURGY
C07C271/20
CHEMISTRY; METALLURGY
C07D213/75
CHEMISTRY; METALLURGY
C07D213/66
CHEMISTRY; METALLURGY
C07D417/04
CHEMISTRY; METALLURGY
C07C271/08
CHEMISTRY; METALLURGY
International classification
C07K1/22
CHEMISTRY; METALLURGY
C07C247/18
CHEMISTRY; METALLURGY
C07D213/75
CHEMISTRY; METALLURGY
Abstract
The present invention relates to linker molecules of formula (1), X-T.sub.b-V.sub.a—U—Y—Z (1) and a method for purifying peptides using said linker molecules. The linker molecule can be coupled to a purification resin via the moiety X and to a peptide via the moiety Y under the release of the leaving group Z. T is an optional spacer moiety and V is an optional electron withdrawing moiety. U is an aryl or 5- or 6-membered heteroaryl moiety bound to at least one electron withdrawing moiety V, W or E. The linker is stable under acidic conditions and releases the peptide upon addition of a reducing agent.
Claims
1. A compound of formula 1, X-Tb-V.sub.a—U—Y—Z (1), wherein X is selected from a moiety of formula 2, 2a, 3, 3a or 4, in particular of formula 2, 2a, 3 or 3a, more particularly of formula 2 or 2a, ##STR00045## wherein each R.sup.1 and R.sup.2 is independently from each other selected from H or B, wherein at least R.sup.1 or R.sup.2 is B, R.sup.3 is selected from H or B, R.sup.4 is selected from H, C.sub.1-C.sub.12-alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid labile protecting group, B is an acid labile amine protecting group, T is a linear or branched spacer comprising at least one, particularly 1 to 5, of the moieties —C.sub.1-12-alkyl-, (C—.sub.2H.sub.4O—).sub.1-12, —C(═O)—, —C(═O)-JR.sup.9—, -JR.sup.9—C(═O)—, -JR.sup.9—, phenyl, 5- or 6-membered heteroaryl, wherein J is CH or N, in particular N, in particular T is a spacer selected from —C.sub.1-C.sub.12-alkyl-, in particular C.sub.1-6-alkyl, more particularly C.sub.1-3-alkyl, —R.sup.5—C(═O)—, —R.sup.5—C(═O)—NR.sup.9—R.sup.6—, —R.sup.5—C(═O)—NR.sup.9—, —C(═O)—NR.sup.9—R.sup.6—, —R.sup.5—NR.sup.9—C(═O)—R.sup.6—, —R.sup.5—NR.sup.9—R.sup.5′—NR.sup.9′C(═O)—R.sup.6—, —R.sup.5—C(═O)—NR.sup.9—R.sup.5′—NR.sup.9′—C(═O)—R.sup.6—, —R.sup.5—NR.sup.9—, —R.sup.5—NR.sup.9—R.sup.6—, —R.sup.5—NR.sup.9—R.sup.5′—NR.sup.9′—R.sup.6—, —R.sup.5—C(═O)—NR.sup.9—R.sup.5′—NR.sup.9′—R.sup.6—, —R.sup.5—C(═O)—O—R.sup.6—, —C(═O)—O—R.sup.6—, —R.sup.5-phenyl-R.sup.6—, —R.sup.5-phenyl-, -phenyl-R.sup.6—, -phenyl-, —R.sup.5-pyrroyl, —R.sup.5-pyrazoyl, —R.sup.5-imidazoyl, R.sup.5-piperazinyl-, —R.sup.5-pyridinyl, —R.sup.5-pyrimidinyl, —R.sup.5-pyrazinyl, —R.sup.5-pyridazinyl, —R.sup.5-pyrroyl-R.sup.6—, —R.sup.5-pyrazoyl-R.sup.6—, —R.sup.5-imidazoyl-R.sup.6—, —R.sup.5-piperazinyl-R.sup.6—, —R.sup.5-pyridinyl-R.sup.6—, —R.sup.5-pyrimidinyl-R.sup.6—, —R.sup.5-pyrazinyl-R.sup.6—, —R.sup.5-pyridazinyl-R.sup.6—, pyrroyl-R.sup.6—, pyrazoyl-R.sup.6—, imidazoyl-R.sup.6-piparazinyl-R.sup.6—, pyridinyl-R.sup.6—, pyrimidinyl-R.sup.6—, pyrazinyl-R.sup.6—, pyridazinyl-R.sup.6—, pyrroyl, pyrazoyl, imidazoyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl and pyridazinyl, wherein R.sup.5, R.sup.5′ and R.sup.6 are independently from each other selected from C.sub.1-C.sub.12-alkyl or (—C.sub.2H.sub.4O—).sub.1-12, in particular C.sub.1-C.sub.6 alkyl, particularly C.sub.1-C.sub.3 alkyl, and wherein R.sup.9 and R.sup.9′ are independently from each other selected from H, C.sub.1-4-alkyl, —C.sub.1-6-alkyl-NH.sub.2, —C.sub.1-6-alkyl-NHB, —C.sub.1-6-alkyl-NB.sub.2, —R.sup.15, —C.sub.1-6-alkyl-R.sup.15, —C.sub.1-6-alkyl-NH—R.sup.15, in particular from H and C.sub.1-2-alkyl, more particularly R.sup.9 is H, wherein B is an independently selected acid labile amine protecting group, R.sup.15 is a blocking agent that is able to react with an aldehyde moiety, in particular R.sup.15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethandithiole, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more particularly cysteinyl, wherein amine and/or thiol moieties of the blocking agent may be protected by an independently selected acid labile amine protecting group B, particularly Boc, and/or an acid labile thiol protecting group, particularly trityl, b is 0 or 1, in particular 1, V is an electron-withdrawing moiety selected from —NR.sup.11—C(═O)—, —C(═O)—NR.sup.11—, —S(═O)—, —NR.sup.12—(CH.sub.2).sub.p—, -piperazinyl-(CH.sub.2).sub.p-, -pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, ##STR00046## —C(═O)—, —C(═O)—O—, in particular —NR.sup.11—C(═O)—, —C(═O)—NR.sup.11—, —S(═O)—, —NR.sup.12—(CH.sub.2).sub.p—, -piperazinyl-(CH.sub.2).sub.p—, -pyridinyl-, pyrimidinyl, more particularly from —NH—C(═O)—, —C(═O)—NH—, —N—(CH.sub.3)—, -piperazinyl-(CH.sub.2).sub.p—, -pyridinyl-, pyrimidinyl, wherein R.sup.11 is selected from H and C.sub.1-4-alkyl, in particular from H and C.sub.1-2-alkyl, more particularly R.sup.11 is H, R.sup.12 is selected from H and C.sub.1-4-alkyl, in particular from H and C.sub.1-2-alkyl, more particularly R.sup.12 is methyl, p is 0, 1 or 2, particularly 0 or 1, a is 0 or 1, wherein the sum of a and b is 1 or 2, U is a phenyl or a five- or six-membered heteroaryl moiety, in particular a phenyl or a six-membered heteroaryl moiety, more particularly a phenyl, that is bound to at least one of the moieties V, W.sub.q and E.sub.n and that may optionally be substituted by C.sub.1-6-alkyl, in particular C.sub.1-3-alkyl, wherein V is defined as described above, W is selected from —N.sub.3, —NO.sub.2, —S(═O)—R.sup.8, —S—S—R.sup.8, —O—CH.sub.2—N.sub.3, —O—C(═O)—O—CH.sub.2—N.sub.3, —N═N-phenyl, —N═N—R.sup.8, ##STR00047## in particular —N.sub.3, —N═N—R.sup.8, —O—CH.sub.2—N.sub.3, —S—S—R.sup.8, wherein R.sup.8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —C.sub.1-C.sub.6-alkyl or —(CH.sub.2).sub.p—NMe.sub.2, in particular pyridyl or —C.sub.1-C.sub.6-alkyl, with p being 1, 2, 3 or 4, E is an electron withdrawing group under acidic conditions, n being is an integer between 0 and 4, in particular 0 and 2, more particularly 0 or 1, and q is an integer between 0 and 4, in particular 0 and 2, more particularly 0 and 1, wherein the sum of n and q is equal or lower than 4, and wherein in case of U being a phenyl moiety and Y being —(CH.sub.2).sub.m—O—C(═O)—, the sum of Hammett constants of V, W, E under acidic conditions is larger than 0.45, and wherein W is in ortho or para position in relation to Y, Y is —(CH.sub.2).sub.m—C(═O)— or —(CH.sub.2).sub.m—O—C(═O)— with m being 1, 2 or 3, in particular 1 or 2, more particularly 1, Z is an electron-withdrawing leaving group.
2. The compound of claim 1, wherein E is selected from piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —N(C.sub.2H.sub.4).sub.2NH.sub.2, —N(C.sub.2H.sub.4).sub.2N—B, —N═N-phenyl, —N═N—R.sup.8, —(CH.sub.2).sub.r—NH—C.sub.1-6-alkyl, —(CH.sub.2).sub.r—N(C.sub.1-6-alkyl).sub.2-, —F, —Cl, —Br, —I, —CN, —NO.sub.2, —N.sub.3, —CF.sub.3, —SO.sub.3H, —CO.sub.2H, —C(═O)NH.sub.2, —SO.sub.2Me, —SOMe, —SO.sub.2Et, —SOEt, in particular pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, —N═N-phenyl, —N═N—R.sup.8, —F, —Cl, —Br, —I, —CN, —NO.sub.2, —N.sub.3, —CF.sub.3, —SO.sub.3H, —CO.sub.2H, more particularly pyridyl, pyrimidinyl, pyridazinyl or —Br with R.sup.8 being pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —C.sub.1-C.sub.6-alkyl or —(CH.sub.2).sub.p—NMe.sub.2, in particular pyridyl or —C.sub.1-C.sub.6-alkyl, with p being 1, 2, 3 or 4, and B being an acid labile amine protecting group, and r being 0, 1, 2, 3 or 4, particularly 0, 1 or 2.
3. The compound according to claim 1, wherein B is selected from Boc (—C(═O)OtBu), Eei (═CMeOEt, 1-ethoxyethylidene) trityl (—C(Ph).sub.3), —C(═O)CPh.sub.3, Mmt (—C(Ph).sub.2C.sub.6H.sub.4OMe), DMT (—C(Ph)(C.sub.6H.sub.4OMe).sub.2), Cbz (—C(═O)OCH.sub.2Ph), benzylideneamine (═CPh), phtalimides (═(CO).sub.2C.sub.6H.sub.4), p-toluenesulfonamides (—SO.sub.2C.sub.6H.sub.4Me), benzylamine (—CH.sub.2Ph), acetamides (—COMe), trifluoroacetamide (—COCF.sub.3), Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), wherein particularly B is Boc or Eei, wherein more particularly B is Boc, and/or the acetal- or ketal protecting groups are selected from ##STR00048## wherein r is 0 to 12, in particular 0 to 6, more particularly 0, 1 or 2, and R.sup.10 is —C.sub.1-C.sub.12-alkyl-, in particular C.sub.1-6-alkyl, more particularly C.sub.1-3-alkyl.
4. The compound according to claim 1, wherein T is selected from —C.sub.1-C.sub.12-alkyl-, in particular C.sub.1-6-alkyl, more particularly C.sub.1-3-alkyl, —R.sup.5—C(═O)—, —R.sup.5—C(═O)—NR.sup.9—, —R.sup.5—NR.sup.9—C(═O)—R.sup.6—, —R.sup.5—C(═O)—NR.sup.9—R.sup.6—, —R.sup.5—C(═O)—NR.sup.9—R.sup.5′—NR.sup.9′C(═O)—R.sup.6—, in particular C.sub.1-3-alkyl, —R.sup.5—C(═O)—NR.sup.9—, —R.sup.5—NR.sup.9—C(═O)—R.sup.6—, more particularly C.sub.1-3-alkyl or —R.sup.5—C(═O)—NR.sup.9—, with R.sup.5, R.sup.5*, R.sup.6, R.sup.9′ and R.sup.9 being as defined above.
5. The compound according to claim 1, wherein the five- or six-membered heteroaryl moiety of U comprises 1 or 2 heteroatoms, in particular the five-membered heteroaryl moiety of the moiety U is selected from pyrazole, imidazole, and the six-membered heteroaryl moiety of the moiety U is selected from pyridine, pyridazine, pyrimidine, pyrazine, particularly pyridine.
6. The compound according to claim 1, wherein U is selected from a moiety of formula 5, 6, 7 or 8, in particular of formula 5 or 6, ##STR00049## wherein T, V, Y, W and E are defined as described above, in case of formula 5 and 6, U is bound to the moiety T or V, in case of formula 7 and 8, U is bound to the moiety V, A.sup.1, A.sup.2, A.sup.3, A.sup.4 and D.sup.1, D.sup.2, D.sup.3, D.sup.4 are independently from each other selected from C, N, S and O, in particular from C and N, wherein 2 to 4 moieties of A.sup.1, A.sup.2, A.sup.3 and A.sup.4 or of D.sup.1, D.sup.2, D.sup.3 and D.sup.4 are C, particularly 3 or 4 moieties of A.sup.1, A.sup.2, A.sup.3 and A.sup.4 or of D.sup.1, D.sup.2, D.sup.3 and D.sup.4 are C, more particularly all moieties A.sup.1, A.sup.2, A.sup.3 and A.sup.4 or of D.sup.1, D.sup.2, D.sup.3 and D.sup.4 are C, n is in case of formulas 5 and 6 an integer between 0 and 3, in particular 0 and 2, in case of formulas 7 and 8 an integer between 0 and 4, in particular 0 and 2, more particular 0 and 1, q is an integer between 0 and 4, in particular 0 and 2, more particular 0 and 1, wherein the sum of n and q is equal or lower than 4.
7. The compound according to claim 5, wherein U is selected from a moiety of formula 9, 10, 11 or 12, in particular of formula 9 or 10, ##STR00050## wherein T, V, Y, W, E, q and n are defined as described above, in case of formula 9 and 10, U is bound to the moiety T or V, in case of formula 11 and 12, U is bound to the moiety V, all moieties A.sup.2, A.sup.3 and A.sup.4 are C or two of A.sup.2, A.sup.3 and A.sup.4 are C and the other two of A.sup.2, A.sup.3 and A.sup.4 is N, in particular A.sup.2 and A.sup.3 are both C, and D.sup.2 is C or N, in particular C.
8. The compound according to claim 1, wherein U is selected from a moiety of formula 13, 14, 15, 16, 17,18, 19, 20 or 21, in particular of formula 13 to 19, more particular of formula 15 or 19, ##STR00051## ##STR00052## wherein T, V, Y, W, E, q and n are defined as described above, in case of formula 13, 14 and 15, U is bound to the moiety T or V, in case of formula 16, 17 and 18, U is bound to the moiety V, A.sup.2, A.sup.3 and A.sup.4 is C or N, in particular C, D.sup.2 is C or N.
9. The compound according to claim 1, wherein Z is selected from: —F, —Cl, —Br, —I, —N.sub.3, —OH, —O(C═O)CH.sub.2(C═O)OH, —SR.sup.14, —OCF.sub.3, —OCH.sub.2CF.sub.3, —OSO.sub.2CF.sub.3, —SO.sub.2C.sub.6H.sub.4CH.sub.3, —SO.sub.2CF.sub.3, —SO.sub.2CH.sub.3 ##STR00053## in particular —OH, —Cl, ##STR00054## in particular —OH, ##STR00055## wherein R.sup.14 is an C.sub.1-C.sub.6-alkyl-, an arylic- or benzylic substituent.
10. A method for purifying peptides comprising the steps of providing a crude linker-modified peptide, wherein the crude peptide is covalently bound to a linker molecule according to claim 1, in a coupling step, coupling the linker-modified peptide with a solid support yielding an immobilized linker-modified peptide, in a releasing step, releasing the peptide, in particular by adding a reducing agent under acidic conditions.
11. The method according to claim 10, wherein in the releasing step, a reduced intermediate characterized by a reduced linker moiety of the immobilized linker-modified peptide is achieved and the peptide is released from said reduced intermediate by a trigger, in particular a change in temperature and/or pH, more particularly by increasing the pH to pH>pKa of the most basic heteroatom of the linker moiety of the reduced intermediate in case of an amine switch, or by decreasing the pH to pH<pKa of the carbamate in case of a carbamate switch.
12. The method according to claim 10, wherein the linker-modified peptide is additionally bound to a synthesis resin and the synthesis resin is cleaved off before the coupling step is performed.
13. The method according to claim 10, wherein the linker molecule comprises a moiety W and/or E that comprises an azide moiety.
14. The method according to claim 10, wherein non-reacted aldehyde moieties of the solid support are blocked using a blocking agent after performing step (b), in particular by using a blocking agent selected from cysteine, threonine, 2-mercaptoethanol, cysteamine, ethandithiole, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, more particularly a blocking agent selected from cysteine and N-methylhydroxylamine.
15. The method according to claim 10, wherein the reducing agent is selected from triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine or tris(2-carboxyethyl)phosphine, trimethyl phosphite, triethyl phosphite, tributyl phosphine, diethyl phosphite, 5,5′-Dithiobis(2-nitrobenzoic acid), sodium dithionite (Na.sub.2S.sub.2O.sub.4), ethandithiole, Propandithiol, dithioerythritol, dithiothreitol, Na.sub.2S, NaSH, glutathione, 2,2′-dithiodipyridine, BH.sub.3, 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, catechol borane, borane tetrahydrofuran, borane dimethyl sulfide, borane dimethylamine complex, borane triphenylphosphine complex, borane tert-butylamine, LiAlH.sub.4, LiBH.sub.4, NaBH.sub.4, NaBH.sub.3CN, NaBH(OMe).sub.3, NaBH(OCCH.sub.3).sub.3, LiAlH(OCMe.sub.3).sub.3, hydroquinone, sodium ascorbate, ascorbic acid, ascorbic acid with KI, hydrazine, NH═NH, formaldehyde, in particular dithioerythritol, dithiothreitol, triphenylphosphine, ascorbic acid with KI, tributylphosphine, trimethylphosphine, tris(2-carboxyethyl)phosphine, sodium dithionite (Na.sub.2S.sub.2O.sub.4), borane dimethyl sulfide, borane triphenylphosphine complex, NaBH.sub.4, ascorbic acid, more particularly triphenylphosphine and trimethylphosphine.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0454]
[0455]
[0456]
[0457]
[0458] General remarks to
[0459] In
[0460]
[0461]
[0462]
[0463] The stability of the linker is still retained after reduction if the pH is higher than the pKa of the carbamate.
[0464]
[0465]
[0466] Example 1 of Linker Type 4: Purification of Naturally Occurring and Research Peptides P1 and P2
[0467] The inventive method for the purification of peptides was applied to two peptides of different polarity, these were H-ARTKQTARKSTGGKA-OH (SEQ ID NO: 1) (P1) fragment 2-16 of the Histone H3 protein and H-AKADEVSLHKWYG-NH.sub.2 (SEQ ID NO: 2) (P2) is a peptide sequence intended for research.
[0468] The peptide sequences were synthesized under standard solid phase peptide synthesis conditions, whereby the synthetic resin was treated with acetic anhydride and pyridine after each amino-acid coupling to block unreacted amino groups. Linker X1 was coupled to P1 on resin by usage of 4 eq. linker, 6 eq. oxyma and 6 eq. diisopropylamine (DIEA) in DMF for 2 h. The inventive method is shown in
[0469] UPLC-MS was used to verify the purity of the individual phases. The UPLC-chromatograms of the non-purified (without linker molecule) and purified peptides are shown in
TABLE-US-00002 TABLE 2 UV- UV-purity sequence purity purified/ calculated vs. No. and linker crude recovery found ESI mass P1 H-ARTKQTAR 41% 93%/62% MH.sup.2+.sub.calc.: 780.95 m/z KSTGGKA-OH MH.sup.2+.sub.found.: 780.63 m/z with X1 P2 H-ARTKQTAR 55% 93%/50% MH.sup.2+.sub.calc.: 780.95 m/z KSTGGKA-OH MH.sup.2+.sub.found.: 780.57 m/z with X2 P1 H-AKADEVSL 41% 95%/73% MH.sup.2+.sub.calc.: 752.89 m/z HKWYG-NH.sub.2 MH.sup.2+.sub.found.: 752.04 m/z with X2
[0470] Purity and recoveries of various peptides after application of the purification process according to the invention
[0471] Example 2 of Linker Type 4: Purification of Naturally Occurring and Research Peptides (P3, P4, P5 and P6)
[0472] In a second set five peptides were synthesized. These were H-YFTGSEVENVSVNVH-NH.sub.2 (SEQ ID NO: 3) (P3) a fragment 81-95 of the human cytomegalovirus lower matrix phosphoprotein (CMV), H-PSNPFYEALST-NH.sub.2 (SEQ ID NO: 4) (P4) fragment 510-520 of humane Lemur Tyrosine Kinase 3 (LMTK3), H-DAEFRHDSGYEVHHQKLVFF-NH.sub.2 (SEQ ID NO: 5) (P5) fragment 1-20 of humane amyloid beta and H-CKADEVSMHKWYG-NH.sub.2 (SEQ ID NO: 6) (P6) a peptide sequence intended for research.
[0473] The peptide sequences P3, P4, P5 and P6 were synthesized in 100 μmol scale under standard solid phase peptide synthesis conditions, whereby the synthetic resin was treated with acetic anhydride and pyridine after each amino-acid coupling to block unreacted amino groups. Linker X1 was coupled to P3, P4, P5 and P6 on resin by usage of 4 eq. linker X1 (301 mg), 6 eq. oxyma (86 mg) and 6 eq. diisopropylamine (DIEA, 105 μL) for 2 h in 1.3 mL DMF. Thereafter, peptides were cleaved of the synthetic resin by a mixture of TFA/PhOH/PhSH/H.sub.2O/ethanedithiol (EDT, 82.5:5:5:2.5) and precipitated in cold diethylether. The crude peptide mixture was dissolved in 4.5 mL Dimethylsulfoxide (DMSO). Aldehyde modified agarose beads (1.5 mL settled beads) were washed each 3× with water and 0.1 M NaCitrate buffer at pH 4.5. To the DMSO solutions of the peptides 10 vol. % (500 μL) of NaCitrate buffer with 8 M guanidium hydrochloride was added. Then the solution was applied to the agarose beads for 90 minutes, what immobilized the desired peptides quantitatively on the agarose beads. Subsequently a 1 w% solution of L-cysteine in 0.1 M NaCitrate buffer at pH 4.5 was added directly to the immobilisation mixture for 15 min to block unreacted aldehyde groups. Afterwards, the purification media was washed each 3× with DMSO, 6 M guanidium hydrochloride, EtOH/water (7:3) with 0.1 M NaCl, water and MeCN to remove any acetylated termination sequences and other impurities. The cleavage of the immobilized linker was carried out by treating the agarose resin with 10 mL of 50 mg per mL PPh.sub.3 MeCN/AcOH/H.sub.2O (90:5:5). Afterwards the support was rinsed 3× with MeCN/H.sub.2O (9:1) and 2 mL of a solution of H.sub.2O/TFA (60:40) was added for 60 min. Thereafter, to the supernatant 2 mL TFA was added and the resulting mixture was filtered into a centrifuge tube and the support was rinsed 2× with TFA/H.sub.2O (95:5) into the same tube. Et.sub.2O was added 5-fold relative to the TFA-water amount to initiate precipitation and the peptide was gained by centrifugation of the tube and disposal of the organic supernatant.
[0474] UPLC-MS was used to verify the purity of the individual phases. The UPLC-chromatograms of the non-purified (without linker molecule) and purified peptides are shown in
TABLE-US-00003 TABLE 3 calculated UV- vs. found sequence purity purified/ UV-purity No. and linker crude recovery ESI mass P3 H-YFTGSEVEN 56% 81%/74%, MH.sup.2+.sub.calc.: 840.41 m/z VSVNVH-NH.sub.2 36 mg MH.sup.2+.sub.found.: 840.50 m/z with X1 P4 H-PSNPFYEAL 78% 91%/76%, MH.sup.2+.sub.calc.: 612.80 m/z ST-NH.sub.2 with 79 mg MH.sup.2+.sub.found.: 612.87 m/z X1 P5 H-DAEFRHDSG 60% 87%/66%, MH.sup.2+.sub.calc.: 820.73 m/z YEVHHQKLVFF- 75 mg MH.sup.2+.sub.found.: 820.94 m/z NH.sub.2 with X1 P6 H-CKADEVSMH 60% 74%/66%, MH.sup.2+.sub.calc.: 776.86 m/z KWYG-NH.sub.2 57 mg MH.sup.2+.sub.found.: 777.05 m/z with X1
[0475] Purity and recoveries of various peptides after application of the purification process according to the invention.
[0476] Example 1 of Type 1 Linker: Purification of Research Peptide P2
[0477] The inventive method for the purification of peptides with a type 1 linker was applied to peptide H-AKADEVSLHKWYG-NH.sub.2 (SEQ ID NO: 2) (P2), which is a peptide sequence intended for research.
[0478] The peptide was synthesized under standard solid phase peptide synthesis conditions, whereby the synthetic resin was treated with acetic anhydride and pyridine after each amino-acid coupling to block unreacted amino groups. Linker X9 of type 1 was coupled to P2 on resin using 4 eq. linker, 6 eq. oxyma and 6 eq. diisopropylamine (DIEA) in dimethylformamide (DMF) for 2 h. Thereafter, the peptide was cleaved off the synthetic resin by a mixture of TFA/TIS/DTT/H.sub.2O (84:2:6:8) and precipitated in diethyl ether. 30 mg of linker modified peptide was gained. The inventive method for this linker type 1 is shown in
TABLE-US-00004 TABLE 4 UV- UV-purity calculated sequence purity purified/ vs. found No. and linker crude recovery ESI mass P2 H-AKADEVSL 77% 48%/ MH.sup.2+.sub.calc.: 752.89 m/z HKWYG-NH.sub.2 45% MH.sup.2+.sub.found.: 752.59 m/z wit hX9
[0479] Purity and recoveries of the peptide after application of the purification process according to the invention
[0480] Example 1 of Type 2 Linker: Purification of Research Peptide P2
[0481] The inventive method for the purification of peptides with a type 2 linker was applied to peptide H-AKADEVSLHKWYG-NH.sub.2 (SEQ ID NO: 2) (P2), which is a peptide sequence intended for research.
[0482] The peptide was synthesized under standard solid phase peptide synthesis conditions, whereby the synthetic resin was treated with acetic anhydride and pyridine after each amino-acid coupling to block unreacted amino groups. Linker X13 was coupled to P2 on resin using 4 eq. linker, 6 eq. oxyma and 6 eq. diisopropylamine (DIEA) in dimethylformamide (DMF) for 3 h. The inventive method of this linker type 2 is shown in
[0483] UPLC-MS was used to verify the purity of the individual steps. The UPLC-chromatograms of the non-purified (without linker molecule) and purified peptide are shown in
TABLE-US-00005 TABLE 5 UV- UV-purity calculated sequence purity purified/ vs. found No. and linker crude recovery ESI mass P2 H-AKADEVSL 77% 97%/ MH.sup.2+.sub.calc.: 752.89 m/z HKWYG-NH.sub.2 73% MH.sup.2+.sub.found.: 752.31 m/z with X13
[0484] Purity and recoveries of the peptide after application of the purification process according to the invention
[0485] Example 1 of Type 3 Linker: Purification of Research Peptide P2
[0486] The inventive method for the purification of peptides with a type 3 linker was applied to peptide H-AKADEVSLHKWYG-NH.sub.2 (SEQ ID NO: 2) (P2), which is a peptide sequence intended for research.
[0487] The peptide was synthesized under standard solid phase peptide synthesis conditions, whereby the synthetic resin was treated with acetic anhydride and pyridine after each amino-acid coupling to block unreacted amino groups. Linker X22 of type 3 was coupled to P2 on resin using 4 eq. linker, 3.6 eq. 2-(6-Chlor-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium-hexafluorophosphat (HCTU), 4 eq. Oxyma and 8 eq. diisopropylamine (DIEA) in dimethylformamide (DMF) for 2 h. Thereafter, the peptide was cleaved off the synthetic resin by a mixture of TFA/TIS/DTT/H.sub.2O (84:2:6:8) and precipitated in diethyl ether. 27 mg of linker modified peptide was gained. The inventive method for this linker type 3 is shown in
TABLE-US-00006 TABLE 6 UV- UV-purity calculated sequence purity purified/ vs. found No. and linker crude recovery ESI mass P2 H-AKADEVSL 77% 26%/ MH.sup.2+.sub.calc.: 752.89 m/z HKWYG-NH.sub.2 20% MH.sup.2+.sub.found.: 752.23 m/z with X22
[0488] Purity and recoveries of the peptide after application of the purification process according to the invention
[0489] Chemical Synthesis of Carbamate Switch (type 4) Linker Molecules X1 and X2
Synthetic Steps for the Synthesis of 2-((2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)carbamoyl)-4-azido-3-bromobenzyl (4-nitrophenyl) carbonate (X1)
6-Amino-7-bromophthalide
[0490] To a cooled solution (0° C.) of 6-aminophthalide (5.13 g, 34.05 mmol) in THF (80 mL) was added N-bromosuccinimide (6.12 g, 34.05 mmol, 1 eq). The cooling bath was removed and the solution stirred for 1 h, then the solvent was removed under reduced pressure. The yellow residue was taken up in ethyl acetate (400 ml) and washed three times with water (200 mL each). The organic phase was dried over magnesium sulfate, filtered and the solvent removed under reduced pressure to give 6-amino-7-bromophthalide as a brown solid (6.53 g, 28.63 mmol, 84%). R.sub.f=0.2 (cyclohexane/ethyl acetate 2:1); UPLC-MS: t.sub.R=1.45 min (gradient 10-90% B in 5 min); UPLC-purity (210 nm)=83.1%; ESI-MS: (calculated MH.sup.+: 227.97, 229.96, found: 228.01, 230.01)
6-azido-7-bromophthalide 6-Amino-5-bromophthalide (5.54 g, 24.17 mmol) was added to 0° C. cold hydrochloric acid (1 M, 100 mL). To the cooled suspension was dropwise added conc. sulfuric acid while stirring until the solid was completely dissolved (25 mL), the solution was further cooled until it reached 0° C. again. A solution of sodium nitrite (3.34 g, 48.34 mmol, 2 eq.) in water (17 mL) was added slowly (formation of nitrous gases if solution too warm). After stirring for 10 minutes, a solution of sodium azide (3.14 g, 48.34 mmol, 2 eq). in water (20 mL) was slowly added dropwise (caution: formation of hydrazoic acid). After 30 min, the suspension was extracted with ethyl acetate (200 mL). The aqueous phase was filtered and the filter cake was washed three times with water (100 mL each) and once with cyclohexane (150 mL). 6-azido-7-bromophthalide (6.58 g (94%), 24.17 mmol, quant.) was obtained as a yellow solid. R.SUB.f.=0.3 (cyclohexane/ethyl acetate 2:1); UPLC-MS: t.SUB.R.=2.24 min (gradient 10-90% B in 5 min); UPLC-purity (210 nm)=50.3%
N-(2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide
[0491] 6-Azido-7-bromophthalide (5.54 g (94%), 24.17 mmol) was taken up in acetonitrile (150 mL) and the suspension heated to 50° C. with stirring. Ethylenediamine (23.4 mL, 350.47 mmol, 14.5 eq.) was added, thus solid completely dissolved after 10 minutes. After 1 h stirring at 50° C., the solvent and excess ethylenediamine were removed under reduced pressure to give a red oil as a residue (8.49 g). Saturated brine (80 mL) was added, the resulting suspension was sonicated for 30 min, stirred at 40° C. for 30 min, and filtered. The filter cake was washed once with saturated brine (50 mL) and once with cyclohexane (100 mL). After drying the filter cake, the title compound was obtained as a yellow solid (3.84 g, 12.2 mmol, 50.6%). Product 3 was also obtained from the filtrate by extraction with ethyl acetate (six times with 150 ml each time) (4.79 g, 15.22 mmol, 63.1%). R.sub.f=0.1 (DCM/MeOH 8:2); UPLC-MS: t.sub.R=1.03 min (gradient 10-90% B in 5 min); UPLC-purity (210 nm)=83.5%; ESI-MS: (calculated MNa.sup.+: 336.01, 338.01 g/mol, found: 335.95, 337.96 m/z).
N-(2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide
[0492] To a stirred solution of bis-(tert-butoxycarbonyl)-(aminooxy)acetic acid ((Boc).sub.2AOAc—OH, 4.71 g, 15.86 mmol, 1.3 eq.) and NHS (1.84 g, 15.86 mmol, 1.3 eq.) in acetonitrile (40 mL) is added dicyclohexylcarbodiimide (DCC, 3.30 g, 15.86 mmol, 1.3 eq.). After stirring for 1 h, the solution is separated from the resulting white precipitate by filtration and the filter cake is washed with acetonitril (40 mL). The filtrate is diluted to 120 mL with acetonitrile. N-(2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2-bromo-6-(hydroxymethyl)benzamide (3.79 g, 12.06 mmol, 1 eq) is taken up in acetonitrile (30 mL) and the suspension is sonicated for 50 min. The filtrate with (Boc).sub.2AOAc-NHS is then added to this suspension and the reaction mixture is stirred for 2.5 hours. After removal of the solvent under reduced pressure, ethyl acetate (150 ml) is added to the resulting orange oil (10.47 g) and the suspension is sonicated for 10 minutes and stirred at 50° C. for 10 minutes. After the suspension was washed, (three times with 80 mL 5 w% NaHCO.sub.3 solution (pH 8), once with 80 mL 2% citric acid solution (pH 4.5) and twice with 80 mL brine), the organic phase was separated and dried with magnesium sulfate and the solvent was removed under reduced pressure, a yellow foam was obtained as a crude product (6.84 g). After drying the crude product under high vacuum, the product was obtained as a yellow solid (6.43 g, 75.89% purity (determined with UV/vis), 8.31 mmol, 68.91% yield). R.sub.f=0.15 (DCM/MeOH 95:5); UPLC-MS: t.sub.R=2.60 min (10-90% MeCN in 3 min), UPLC-purity (21 nm)=79.1%, ESI-MS: (calculated MNa.sup.+: 609.13, 611.13 g/mol, found: 609.03, 611.06 m/z).
[0493] 2-((2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)carbamoyl)-4-azido-3-bromobenzyl (4-nitrophenyl) carbonate
[0494] N-(2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2-bromo-6-(hydroxymethyl) benzamide (6.39 g (79%), 8.26 mmol) was dissolved in DCM (20 mL) and cooled to 0° C. To the solution was first added anhydrous pyridine (1.00 mL, 12.48 mmol, 1.5 eq.) with stirring and then slowly a solution of p-nitrophenyl chloroformate (2.52 g, 12.48 mmol, 1.5 eq.) in DCM (20 mL). The reaction mixture was warmed to room temperature and stirred for 1 h. Under reduced pressure, the solvent is removed and the resulting orange oil (9.99 g) is dissolved in 150 mL ethyl acetate. The suspension was filtered, and the solvent was removed from the filtrate under reduced pressure to give a yellow foamy solid as a crude product (8.85 g). After purification by column chromatography (silica gel, cyclohexane: ethyl acetate 2:1 to 1:1), the product (3.82 g) was taken up in 100 mL diethyl ether, treated with ultrasound for 10 min, stirred for 30 min at 40° C. and then overnight at −20° C. stored. The product was filtered and washed with 100 mL −20° C. cold diethyl ether and dried after high vacuum as a pale-yellow solid (2.47 g, 3.29 mmol, 39.5%).
[0495] R.sub.f=0.25 (Ethyl acetate/Cyclohexane 2:1), UPLC-MS: t.sub.R=3.18 min (10-90% MeCN in 5 min), UPLC-purity (278 nm)=88.4%, ESI-MS: (calculated MNa.sup.+: 774.13, 776.13 g/mol, found: 773.91, 775.88 m/z).
[0496] .sup.1H NMR (500 MHz, DMSO) δ 8.71 (s, 1H), 8.32 (d, J=9.2 Hz, 2H), 7.95 (s, 1 H), 7.62 (d, J=8.3 Hz, 1H), 7.57 (d, J=9.2 Hz, 1H), 7.52 (d, J=8.3 Hz, 1H), 5.25 (s, 2H), 4.36 (s, 2H), 3.33 (m, 4H), 1.46 (s, 18H).
Synthetic steps for the synthesis of 2-((2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)carbamoyl)-4-azido-3,5-dibromobenzyl (4-nitrophenyl) carbonate (X2)
5,7-Dibromo-6-aminophthalide
[0497] 6-Aminophthalide (20.00 g, 132.75 mmol) was provided in a 1 L round bottom flask with a stirring bar and 550 mL THF and 30 mL MeCN was added at 0° C. To the solution N-bromosuccinimide was added slowly as a solid through a powder funnel after witch the solution turned brownish. The ice-bath was removed then after some time the solution turned yellow. After 2 h stirring at room-temperature UPLC-MS and TLC indicated complete conversion to the dibromide. The solvent was removed under reduced pressure at a rotational evaporator. The remaining solid was dissolved in 600 ml Ethyl acetate and washed 3 times with water. The organic phase was dried with MgSO.sub.4 and after evaporation 39.86 g (129.86 mmol, 98%) of the desired product was gained as a pale yellow solid. R.sub.f=0.6 (cyclohexanes/Ethyl acetate 2:1), UPLC-MS: t.sub.R=2.36 min (10-90% MeCN in 3 min), UPLC-purity (254 nm)=87.0%, ESI-MS: (calculated MH.sup.+: 307.95 g/mol, found: 307.76 m/z).
5, 7-Dibromo-6-azidophthalide
[0498] 5,7-Dibromo-6-aminophthalide (38.50 g, 124.18 mmol) was dissolved in 200 mL concentrated H.sub.2SO.sub.4 in a 2 L flask, the brown solution was cooled with a big ice bucket then it was slowly added 235 mL 1 M HCl, a precipitate formed during HCl addtion. NaNO.sub.2 (17.31 g, 248.35 mmol, 2 eq) was dissolved in 32 mL water and was added slowly to the suspension after it reached 5° C. The precipitate dissolved thereafter. The solution was further stirred for 15 min at 0° C. and subsequently, NaN.sub.3 (16.31 g, 248.35 mmol, 2 eq) in 75 mL water was added dropwise by Pasteur pipette. Strong formation of gases (N.sub.2, HN.sub.3) was observed. The foamy solution was stirred for 1 h, thereafter 500 ml water was added under cooling with ice. After foam-building had ceased and solution had reached room-temperature the suspension was filtered by the use of a Büchner funnel, while 2 L of water was used to transfer the solid into the funnel and thereby wash the solid. After drying the wet product in a crystallizing dish under reduced pressure, the product was yielded as a slightly brownish solid (36.01 g, 108.16 mmol, 87.1%). R.sub.f=0.45 (cyclohexanes/Ethyl acetate 2:1), UPLC-MS: t.sub.R=2.89 min (10-90% MeCN in 3 min), UPLC-purity (254 nm)=95.3%,
N-(2-aminoethyl)-3-azido-2, 4-dibromo-6-(hydroxymethyl)benzamide
[0499] 5,7-Dibromo-6-azidophtalide (18.00 g, 53.52 mmol) was dissolved in Ethyl acetate (490 mL) insoluble impurities were filtered off and ethylenediamine (52.73 mL, 749.32 mmol, 14 eq) was added at 0° C. The reaction was stirred at room temperature for 1 h, after which UPLC-MS showed quantitative conversion. The reaction mixture was transferred into a separation funnel to which 100 mL Brine was added. After separation of the aqueous phase the organic phase was dried with MgSO.sub.4 and the desired product was gained after evaporating the organic solvent on rotational evaporator as an orange solid. (20.50 g, 52.16 mmol, 97.4%). R.sub.f=0.25 (DCM/MeOH 8:2), UPLC-MS: t.sub.R=1.80 min (10-90% MeCN in 3 min), UPLC-purity (254 nm)=84.2%, ESI-MS: (calculated MH.sup.+: 393.93 g/mol, found: 393.87 m/z).
N-(2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2,4-dibromo-6-(hydroxymethyl)benzamide
[0500] Bis-(tert-Butoxycarbonyl)-(aminooxy)acetic acid ((Boc).sub.2AOAcOH, 17.30 g, 58.18 mmol, 1.1 eq.) and N-hydroxysuccinimide (NHS, 6.76 g, 58.18 mmol, 1.1 eq.) was dissolved in 350 mL Acetonitril. To this solution was added dicyclohexylcarbodiimide (DCC, 12.13 g, 58.18 mmol, 1.1 eq.) as a solid, after dissolution of DCC a white precipitate formed. The reaction mixture was stirred for 1 h at room temperature where the (Boc).sub.2AOAc-NHS ester was quantiatively formed according to UPLC-MS. Thereafter the mixture was filtered into a 1 L flask to remove the DCC-urea. N-(2-aminoethyl)-3-azido-2,4-dibromo-6-(hydroxymethyl)benzamide (20.5 g, 52.90 mmol) was dissolved in 530 mL Ethyl acetate and subsequently added to the solution of (Boc).sub.2AOAc-NHS. The mixture was stirred at room temperature for 1 h after which the completion of the reaction was confirmed by TLC and UPLC-MS. Additional formed precipitate was filtered off and the organic phase was washed 2× with 5% NaHCO.sub.3 (each 200 mL), 1× brine and 2×2% citric acid solution(pH 4.5)/brine 1:1 (each 150 mL). The organic phase was dried with MgSO.sub.4 and hence, the organic solvent was removed in vacuo and the title compound was obtained as a pale yellow oil (37.70 g, 56.58 mmol, quantitativ). R.sub.f=0.4 (DCM/MeOH 95:5), UPLC-MS: t.sub.R=2.97 min (10-90% MeCN in 3 min), UPLC-purity (254 nm)=54.2%, ESI-MS: (calculated MH.sup.+: 667.05, MNa.sup.+: 689.04 g/mol, found: 688.95 m/z).
2-((2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)carbamoyl)-4-azido-3,5-dibromobenzyl (4-nitrophenyl) carbonate
[0501] N-(2-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetamido)ethyl)-3-azido-2,4-dibromo-6-(hydroxymethyl)benzamide (37.70 g (94%), 52.90 mmol was provided in CH.sub.2Cl.sub.2 (170 mL) and (dry, stored over molecular sieve) pyridine (4.72 mL, 58.50 mmol, 1.1 eq) was added. Thereafter, 4-nitrophenylchloroformiate (12.03 g, 58.50 mmol, 1.1 eq) was added slowly as a solid at room temperature keeping temperature constant by usage of a water bath. Reaction may cause DCM to evaporate at the center of the flask. Indicated by LCMS and TLC indicated a complete reaction after 1 h. Dichloromethane was removed in vacuo yielding 52 g crude brown oil and the residue was dissolved in 500 mL ethyl acetate and washed 2×2% citric acid solution (pH 4.5)/brine 1:1 (each 250 mL) and 1× brine 150 mL. The organic phase was dried by usage of MgSO.sub.4. Afterwards this suspension was filtered over a 50 g silica plug in a glas frit, whereas the orange and reddish impurities remained on silica. The organic solvent was removed from the filtrate under reduced pressure till a highly viscous slightly amber oil remained. To this oil 70 ml of Et.sub.2O was added and the two-phasic emulsion was turned on a rotational evaporator at 45° C. for 10 min till one homogeneous phase was formed. A small sand corn was added to the flask as a crystallization initiator and the flask was put in a refrigerator overnight (16 h). In the flask a sluggish precipitate had formed. Further 200 mL of cold (-25° C.) ether was added to the flask and the flask was gently shaken and stirred in an ice-bath. The white star-like crystals were transferred and washed with additional 200 mL cold Et.sub.2O into a paper filter filled Büchner-funnel. Thus, yielding the title compound as a white solid (29.95 g, 36.02 mmol, 68.1%). R.sub.f=0.6 (Ethyl acetate/Cyclohexane 2:1), UPLC-MS: t.sub.R=2.86 min (30-95% MeCN in 3 min), UPLC-purity (278 nm)=93.5%, ESI-MS: (calculated MH.sup.+: 832.06, MNa.sup.+: 854.04 g/mol, found: 853.87 m/z).
[0502] .sup.1H NMR (400 MHz, DMSO) δ 8.73 (s, 1H), 8.33 (d, J=9.1 Hz, 2H), 7.93 (s, 1 H), 7.57 (d, J=9.1 Hz, 2H), 5.24 (s, 2H), 4.36 (s, 2H), 3.40-3.32 (m, 4H), 1.46 (s, 18H).
[0503] Chemical Synthesis of Amine Switch with Azide Reductive Safety Lock (Type 1) Linker Molecules X6 and X9
Synthetic Steps for the Synthesis of 3-((4-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetyl)piperazin-1-yl)methyl)-4-azidobenzyl(4-nitrophenyl) carbonate (X6)
4-Nitro-3-(piperazin-1-ylmethyl)benzoic acid
[0504] 4-Nitro-3-methylbenzoic acid methyl ester (5.03 g, 25.50 mmol) was dissolved in 170 mL dry Benzene in a 500 mL round bottom flask with a stirring bar. N-Bromosuccinimide (5.27 g, 29.33 mmol) and Benzoyl peroxide (0.62 g, 2.55 mmol) were added and the solution was heated to reflux. After 12 h UPLC-UV/vis indicated only 10% conversion to the brominated starting material. Again, N-Bromosuccinimide (3.66 g, 20.56 mmol) and Benzoyl peroxide (0.62 g, 2.55 mmol) were added. After 36 h UPLC-UV/vis indicated a conversion of 80%. The mixture was concentrated in vacuo and 85 mL Chloroform was added. It was slowly filtrated to a mixture of Piperazine (8.87 g, 102.00 mmol) and K.sub.2O0.sub.3 (4.63 g, 33.15 mmol) in 85 mL Chloroform in a 500 mL round bottom flask with a stirring bar. After 2 h UPLC-UV/vis indicated complete conversion to the desired product. With a rotational evaporator the solvent was removed. To the residue was added 250 mL Ethyl acetate and the mixture was filtrated to a separation funnel. The organic phase was washed three times with 100 mL saturated NaHCO.sub.3 and three times with 100 mL Brine. After drying the organic phase over MgSO.sub.4 and evaporation 7.3 g (not completely dry) of the desired product was obtained as a yellow oil. UPLC-MS: tR=1.602 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=68.3%, ESI-MS: (calculated MH+: 280.12 g/mol, found: 280.11 m/z).
(4-nitro-3-(piperazin-1-ylmethyl)phenyl)methanol
[0505] 4-Nitro-3-(piperazin-1-ylmethyl)benzoic acid (1.10 g, assumed 3.00 mmol) was dissolved in 8 mL THF in a 250 mL round bottom flask and stirred at RT with a magnetic stirring bar. LiCl (0.77 g, 18.00 mmol), NaBH4 (0.69 g, 18.00 mmol) and 16 mL Ethanol were added successively. After 12 h UPLC-UV/vis indicated complete conversion of the starting material. The reaction mixture was concentrated in vacuo and the residue was suspended in 60 mL Ethyl acetate. While stirring the mixture rapidly 25 mL 1 M NH.sub.4Cl was added dropwise. After 2 h 25 mL 1 M NaOH was added slowly and the mixture was transferred to a separation funnel. The layers were separated, and the aqueous phase was extracted twice with 50 mL Ethyl acetate and twice with 50 mL Chloroform. The combined organic layers were dried over MgSO4 and after evaporation of the organic solvent the desired product was obtained as a yellow solid (0.59 g, 2.35 mmol, 88%). UPLC-MS: tR=1.27 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=50.4%, ESI-MS: (calculated MH+: 252.29 g/mol, found: 252.17 m/z).
2-bis-(tert-Butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrobenzyl)piperazin-1-yl)ethan-1-one
[0506] In a 100 mL round bottom flask Bis-Boc-protected Aminooxy acetic acid (0.78 g, 2.63 mmol) and N-Hydroxysuccinimide (0.31 g, 2.63 mmol) were dissolved in 25 mL Acetonitrile and stirred with a magnetic stirring bar. Dicyclohexylcarbodiimide (0.55 g, 2.63 mmol) was added and the mixture was stirred for 1.5 h at RT. After filtration to a dropping funnel it was slowly added to a solution of 4-Nitro-3-(piperazin-1-ylmethyl)benzyl alcohol (0.59 g, 2.35 mmol) in 25 mL Chloroform in a 250 mL round bottom flask. After 1 h UPLC-UV/vis indicated complete conversion of the starting material. The solvent was removed with a rotational evaporator and the residue was suspended in 100 mL Ethyl acetate and transferred to a separation funnel. The organic phase was washed three times with 50 mL water, three times with 50 mL saturated NaHCO3-solution and three times with 50 mL Brine and was dried over MgSO.sub.4. The desired product was obtainend after evaporation of the organic solvent as a yellow solid (1.8 g, not completely dry). UPLC-MS: tR=2.24 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=19.8%, ESI-MS: (calculated MH+: 525.14 g/mol, found: 525.25 m/z)
1-(4-(2-amino-5-(hydroxymethyl)benzyl)piperazin-1-yl)-2-bis-(tert-Butoxycarbonyl)-(aminooxy)ethan-1-one
[0507] In a round bottom flask 2-bis-(tert-Butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrobenzyl)piperazin-1-yl)ethan-1-one was dissolved in 16 mL water/ethanol (1:4). After this iron powder (0.23 g, 4 mmol) and ammonium chloride (0.25 g, 4 mmol) were added to the solution. The reaction mixture was stirred over night at room temperature. After confirming full consumption of the startmaterial via UPLC-MS the reaction mixture was filtered over celite to remove excess iron. The solvent was concentrated as far as possible in vacuo. After adding 50 mL of CHCl.sub.3 the organic phase was washed with 3×50 mL sat. NaHCO.sub.3 solution, 3×50 mL Brine, dried over MgSO.sub.4 and the organic solvent removed under reduced pressure at a rotational evaporator. The final product (0.19 g, 0.4 mmol) was obtained as a brown oil. UPLC-MS: t.sub.R=2.05 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=24.7%, ESI-MS: (calculated MH.sup.+: 495.28, MNa.sup.+: 517.26 g/mol, found: 517.29 m/z).
2-bis-(tert-Butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)benzyl)piperazin-1-yl)ethan-1-one
[0508] In a round bottom flask 1-(4-(2-amino-5-(hydroxymethyl)benzyl)piperazin-1-yl)-2-bis-(tert-Butoxycarbonyl)-(aminooxy)ethan-1-one (0.19 g, 0.4 mmol) was dissolved in dry Acetonitrile. The solution was cooled via ice bath while under stiring tert-butyl nitrile (236 μL, 2 mmol), and then trimethylsilyl azide (351 μL, 1.6 mmol) were slowly added. The solution was further stirred with ice cooling for 2 h in a sealed flask. After UPLC-MS showed incomplete conversion tert-butyl nitrile (572 μL, 4 mmol), and then trimethylsilyl azide (702 μL, 3.2 mmol) were slowly added again. The reaction mixture was stirred further for 2 h. The solvent was then removed under reduced pressure at a rotational evaporator. After dissolving the crude product in 50 mL ethyl acetate the organic phase was washed with 3×50 mL NaHCO.sub.3, 3×0 50 mL Brine solution and dryed with Magnesiumsulfate. The desired product (0.14 g, 0.20 mmol) was gained after evaporating the organic solvent. UPLC-MS: t.sub.R=2.34 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=20.4%, ESI-MS: (calculated MH.sup.+: 521.27, found: 521.30 m/z).
3-((4-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetyl)piperazin-1-yl)methyl)-4-azidobenzyl (4-nitrophenyl) carbonate (X6)
[0509] 2-bis-(tert-ButoxycarbonyI)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)benzyl)piperazin-1-yl) ethan-1-one (149 mg, 0.2 mmol) was dissolved in a round bottom flask using 0.2 mL of dry DCM. Pyridin (19.2 μL, 0.24 mmol) was added and the solution was cooled using an ice bath. To this mixture a solution of p-nitrophenylchloroformiate (32.9 mg, 0.16 mmol) in 0.2 mL of dry DCM was slowly added and the reaction mixture was stirred overnight at room temperature. The organic solvent was removed in vacuo and the desired product was obtained as a dark brown oil (0.21 g, not completely dry) UPLC-MS: t.sub.R=10.72 min (0-60% MeCN in 11 min), UPLC-purity (278 nm)=8.39%, ESI-MS: (calculated MH.sup.+: 686.28, MNa.sup.+: 708.26 g/mol, found: 686.38 m/z).
Synthetic Steps for the Synthesis of 3-(4-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetyl)piperazin-1-yl)-4-azidobenzyl(4-nitrophenyl) carbonate (X9)
1-(4-(2-amino-5-(hydroxymethyl)phenyl)piperazin-1-yl)-2-bis-(tert-Butoxycarbonyl)-(aminooxy)ethan-1-one
[0510] 2-bis-(tert-Butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazin-1-yl)ethan-1-one (1.00 g, 1.96 mmol) was dissolved in 78 mL of Methanol in a round bottom flask. To this solution Magnesium powder (1.28 g, 19.6 mmol) was added. Under stiring then ammonium formiate (1.23 g, 19.6 mmol) was added as a solid. The solution was further stirred for 20 min at room temperature while formation of gases was observed. The solution was filtered off immediately to remove Magnesium and the solvent was removed under reduced pressure at a rotational evaporator. The crude product was freeze dryed using a solvent mixture of 1:1 water/Acetonitrile. The crude product (2.20 g) was used without any further purification. UPLC-MS: t.sub.R=2.21 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=53.2%, ESI-MS: (calculated MH.sup.+: 481.27 g/mol, MNa.sup.+: 503.25 g/mol, found: 477.30 m/z).
2-bis-(tert-Butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)phenyl)piperazin-1-yl)ethan-1-one
[0511] In a round bottom flask 1-(4-(2-amino-5-(hydroxymethyl)phenyl)piperazin-1-yl)-2-bis-(tert-Butoxycarbonyl)-aminooxy)ethan-1-one (1.00 g, 2.08 mmol) was dissolved in dry Acetonitrile. The solution was cooled via ice bath while under stiring tert-butyl nitrile (1.37 mL, 10.4 mmol), and then trimethylsilyl azide (1.16 mL, 8.32 mmol) were slowly added. The solution was further stirred with ice cooling for 2 h in a sealed flask. After UPLC-MS showed incomplete conversion Tert-butyl nitrile (1.37 mL, 10.4 mmol) and then trimethylsilyl azide (1.16 mL, 8.32 mmol) were slowly added again. The reaction mixture was stirred further overnight. The solvent was then removed under reduced pressure at a rotational evaporator. After dissolving the crude product in 50 mL ethyl acetate the organic phase was washed with 3×50 mL NaHCO.sub.3, 3×50 mL Brine solution and dryed with Magnesiumsulfate. The desired product (0.2 g, 0.39 mmol) was gained after evaporating the organic solvent and purification via flash column (3:2 EtOAC/Cyclohexane). R.sub.f=0.5 (EtOAc/Cyclohexane 4:1), UPLC-MS: t.sub.R=2.92 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=84.2%, ESI-MS: (calculated MH.sup.+: 507.26 g/mol, MNa.sup.+: 529.24 g/mol, found: 529.30 m/z).
3-(4-(2-bis-(tert-Butoxycarbonyl)-(aminooxy)acetyl)piperazin-1-yl)-4-azidobenzyl(4-nitrophenyl) carbonate (X9)
[0512] 2-bis-(tert-Butoxycarbonyl)-(aminooxy)-1-(4-(2-azido-5-(hydroxymethyl)phenyl)piperazin-1-yl)ethan-1-one (76 mg, 0.15 mmol) was dissolved in a round bottom flask using 1 mL of dry DCM. Pyridin (12.2 μL, 0.15 mmol) was added and the solution was cooled using an ice bath. To this solution p-nitrophenylchloroformiate (20.3 mg, 0.15 mmol) was slowly added as a solid. After 10 min the ice bath was removed and the reaction mixture was stirred overnight at room temperature. The organic solvent was removed in vacuo and the crude product was re-dissolved in 30 mL EtOAc. The organic phase was then washed with 3×sat. NaHCO.sub.3 solution, 3×Brine, dryed over MgSO.sub.4 and the organic solvent again removed in vacuo. The crude product was finally purified via flash column using a EtOAc/Cyclohexane (1:1) solvent mixture. The desired compound was obtained as a pale yellow oil (30 mg, 0.05 mmol). R.sub.f=0.54 (EtOAC/Cyclohexane 1:1), UPLC-MS: t.sub.R=3.53 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=93.6%, ESI-MS: (calculated MH.sup.+: 671.66, MNa.sup.+: 694.24 g/mol, found: 694.40 m/z).
[0513] .sup.1H-NMR (400 MHz, CDCl.sub.3, 25° C.): δ [ppm]=8.28 (d, J=9.0 Hz, 2H), 7.38 (d, J=9.0 Hz, 2H), 7.22-7.06 (m, 3H), 5.22 (s, 2H), 4.62 (s, 2H), 3.94 (t, J=3.93, 2H), 3.81 (t, J=3.81, 2H), 3.12 (t, J=3.12, 2H), 3.07 (t, J=3.07, 2H), 1.54 (s, 18H).
[0514] Chemical Synthesis of Amine Switch with Other Reductive Safety Lock (Type 2) Linker Molecules X12, X13 and X43
Synthesis of (5-(2-(2-bis-(tert-Butoxycarbonyl(aminooxy)acetamido)-6-(tert-butyldisulfanyl)pyridin-3-yl)methyl (4-nitrophenyl) carbonate (X12)
Synthesis of methyl 6-meracapto-5-nitropyridine-3-carboxylate
[0515] To a cooled solution of methyl 6-chloro-5-nitropyridine-3-carboxylate (1.6 gm, 7.037 mmol) in methanol (25 ml) 70% sodium hydrosulfide hydrate (1.115, 14.127 mmol) was added in small portions. The mixture stands with stirring for 30 min-1 h. Subsequently, the solid material was filtered. The remining solution was reduced to 5 ml using rotatory evaporator. The remining solution was acidified to pH 2 by slow addition of 1M HCl at 0° C. The resulting yellow solid material was collected by filtration and the material is used for further steps with out purification. UPLC-MS: t.sub.R=1.80 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=95.0%, ESI-MS: (calculated MH.sup.+: 215.01 g/mol, found: 215.20 m/z.
Synthesis of methyl 6-meracapto-5-aminopyridine-3-carboxylate methyl 6-meracapto-5-nitropyridine-3-carboxylate (1.00 gm, 4.537 mmol) and 1.85 gm (32.48 mmol) of iron powder were placed in the reaction flask containing 50 mL of 75% Methanol and 25% water. Calcium chloride (0.41 gm, 3.63 mmol) was then added and the mixture was refluxed on oil bath till the starting material completely converts to the product. At the end of the reflux period, the mixture was filtered celite to remove excess iron. The filtrate was concentrated to near dryness and subsequently water (25 ml) was added, and compound was extracted using ethyl acetate (3×25 ml). The solvent was removed under reduced pressure and the obtained crude residue was pure enough to proceed further (3.49 mmol, 77%). UPLC-MS: t.SUB.R.=1.26 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=93%, ESI-MS: (calculated MH.SUP.+.: 185.04 g/mol, found: 185.14 m/z).
Synthesis of (5-amino-6-m eracaptopyridin-3-yl)methanol
[0516] Methyl 6-meracapto-5-aminopyridine-3-carboxylate (0.7 gm, 3.68 mmol) was dissolved in dry THF (20 mL) and the solution was cooled to 0° C. Lithium aluminum hydride (1.42 gm, 36.89 mmol) was added portion wise to the reaction mixture under N.sub.2 atmosphere during 10 min. The resulting solution was stirred for 24 h at room temperature. After completion of the reaction as indicated by UPLC, the excess of LiAlH.sub.4 was quenched by adding simultaneously 1.4 ml of water, 1.4 ml 10% NaOH, 4.2 ml of water at 0° C. Al salts were filtered off and the solid materials washed with water and MeOH (1:1, 100 ml). The solvent was evaporated, and the compound was extracted from solid cake using isopropanol (3×50 ml). Isopropanol was removed under reduced pressure to obtain desired product (1.92 mmol, 52%) in pure form. UPLC-MS: t.sub.R=1.01 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=96%, ESI-MS: (calculated MH.sup.+: 157.04 g/mol, found: 157.11 m/z)
Synthesis of (2-(2-bis-(tert-Butoxycarbonyl)(aminooxy))-N-(2-(tert-butyldisulfanyl)-5-(hydroxymethyl)pyridin-3-yl) acetamide)
[0517] To a Schlenk flask charged with 6-mercapto-5-amino pyridine-3-carboxylic acid (1.3 gm, 8.03 mmol) was added CH.sub.2Cl.sub.2 (20 mL), 2-Methyl-2-propanethiol (0.91 ml, 8.03 mmol) and TBHP (1.13 gm, 8.83 mmol, 70% solution in water) under N.sub.2 atmosphere. After 30 seconds, NIS (0.19 gm, 0.18 mmol) was added in one batch. Then the Schlenk flask was allowed to react for 1 h at 25° C. After the completion of the reaction, it was quenched by water (20.0 mL) and extracted with ethyl acetate (3×30 mL). The organic layers were combined and evaporated under vacuum and the compound was used in next steps without further purification. UPLC-MS: t.sub.R=1.68 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=93%, ESI-MS: (calculated MH.sup.+: 245.08 g/mol, found: 245.18 m/z).
Synthesis of (5-(2-(2-bis-(tert-Butoxycarbonyl)(aminooxy)acetamido))-6-(tert-butyldisulfanyl)pyridin-3-yl)methyl (4-nitrophenyl) carbonate (X12)
[0518] (5-amino-6-(tert-butyldisulfanyl)pyridin-3-yl)methanol (83.5 mg, 0.156 mmol mmol) was provided in CH.sub.2Cl.sub.2 (2 mL) and dry, pyridine (0.161 mL, 0.19 mmol) was added. Thereafter, 4-nitrophenylchloroformiate (31.9 mg, 0.154 mmol) was added. Indicated by LCMS and TLC reaction had complete after 12 h. Dichloromethane was removed in vacuo yielding a pale yellow/orange crude oil and the residue was re-dissolved in 10 mL ethyl acetate and washed water and brine 1:1 (each 5 mL). Finally EtoAc was removed under reduced pressure to obtain desired product (0.08 mmol, 75%). UPLC-MS: tR=12.59 min (10-60% MeCN in 11.50 min, 60-90% 11.51-13 min), UPLC-purity (=nm)=70%, ESI-MS: (calculated MNa+: 705.19 g/mol, found: 705.41 m/z).
Synthetic Steps for the Synthesis of 3-(4-(2-bis-(tert-Butoxycarbonyl)-((aminooxy)acetyl)piperazin-1-yl)-4-nitrobenzyl (4-nitrophenyl) carbonate (X13)
Methyl 3-fluoro-4-nitrobenzoate
[0519] 3-Fluoro-4-nitrobenzoic acid (5.00 g, 27.01 mmol) was dissolved in 100 mL Methanol in a 250 mL round bottom flask with a stirring bar and 3 mL (54,02 mmol) H.sub.2SO.sub.4was added. After 42 h stirring at 50° C. UPLC-UV/vis indicated complete conversion to the methyl ester. With a rotational evaporator the reaction mixture was concentrated to 10 mL. After addition of 100 mL Ethyl acetate and 100 mL H.sub.2O the mixture was stirred and subsequently K.sub.2O0.sub.3 (7.00 g) was added portion wise. The mixture was transferred to a separation funnel, the layers were separated and the aqueous phase was extracted three times with 100 mL Ethyl acetate. The combined organic layers were dried over MgSO.sub.4 and after evaporation 4.81 g (24.15 mmol, 89%) of the desired product was gained as an orange solid. UPLC-MS: t.sub.R=2.53 min (10-90% MeCN in 3 min), UPLC-purity (210 nm)=99.4%, ESI-MS: (calculated MH.sup.+: 200.14 g/mol, found: −).
Methyl 4-nitro-3-(piperazin-1-yl)benzoate
[0520] Piperazine (4.07 g, 47.20 mmol) and K.sub.2CO.sub.3 (4.24 g, 30.68 mmol) were suspended in 50 mL Chloroform in a 250 mL round bottom flask with a stirring bar. methyl 3-fluoro-4-nitrobenzoate (4.7 g, 23.60 mmol), dissolved in 50 mL Chloroform, was added slowly via dropping funnel at RT over 1 h and the mixture was stirred rapidly. After 66 h UPLC-UV/vis indicated that only 10% of the starting material was conversed. Piperazin (4.07 g, 47.20 mmol) was added and after 2 h UPLC-UV/vis indicated a complete conversion. After 15 minutes 100 mL Chloroform and 150 mL saturated NaHCO.sub.3-solution were added, and the mixture was transferred to a separation funnel. The layers were separated, and the organic phase was washed twice with 100 mL saturated NaHCO.sub.3-solution and once with 100 mL Brine. After drying over MgSO.sub.4 the desired product was obtained after evaporating the organic solvent on rotational evaporator as a red solid (7.87 g, not completely dry). UPLC-MS: t.sub.R=1.72 min (10-90% MeCN in 3 min), UPLC-purity (210 nm)=87.2%, ESI-MS: (calculated MH.sup.+: 266.27 g/mol, found: 266.23 m/z).
(4-nitro-3-(piperazin-1-yl)phenyl)methanol
[0521] methyl 4-nitro-3-(piperazin-1-yl)benzoate (5.85 g, not completely dry, assumed 20.00 mmol) was dissolved in 40 mL THF in a 500 mL round bottom flask and stirred with a magnetic stirring bar. LiCI (5.09 g, 120.00 mmol), NaBH.sub.4 (4.54 g, 120.00 mmol) and 80 mL Ethanol were added successively. After 15 h UPLC-UV/vis indicated a complete conversion. The reaction mixture was concentrated in vacuo, 20 mL Chloroform was added, and the mixture was stirred rapidly while 30 mL of a 2 M NH.sub.4Cl solution was added dropwise. After 1 h 120 mL 1 M NaOH and 80 mL Chloroform were added slowly, and the mixture was transferred to a separation funnel. The layers were separated, and the aqueous phase was extracted three times with 100 mL Chloroform. The combined organic layers were dried over MgSO.sub.4 and after evaporation of the organic solvent the desired product was obtained as an orange solid (3.97 g, 16.73 mmol, 84%). UPLC-MS: t.sub.R=1.27 min (10-90% MeCN in 3 min), UPLC-purity (210 nm)=70.0%, ESI-MS: (calculated MH.sup.+: 238.26 g/mol, found: 238.24 m/z).
2-bis-(tert-Butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazin-1-yl)ethan-1-one
[0522] Bis-(tert-Butoxycarbonyl)-(aminooxy)acetic acid ((Boc).sub.2AOAcOH, 2.96 g, 9.96mmo1, 1.2 eq.) and N-hydroxysuccinimide (NHS, 1.15 g, 9.96 mmol, 1.2 eq.) was dissolved in 10 mL Acetonitril. To this solution was added dicyclohexylcarbodiimide (DCC, 2.07 g, 9.96 mmol, 1.2 eq.) as a solid, after dissolution of DCC a white precipitate formed. The reaction mixture was stirred for 1 h at room temperature where the (Boc).sub.2AOAc-NHS ester was quantitatively formed according to UPLC-MS. Thereafter the mixture was filtered into a round bottom flask where (4-nitro-3-(piperazin-1-yl)phenyl)methanol (1.98 g, 8.30 mmol) was provided in 83 mL of Chloroform. The mixture was stirred at room temperature for 1 h after which the completion of the reaction was confirmed by TLC and UPLC-MS. Additional formed precipitate was filtered off and the organic phase was washed 3×water, 3×sat. NaHCO3 and 3×brine solution. The organic phase was dried with MgSO.sub.4 and hence, the organic solvent was removed in vacuo and the title compound was obtained as a yellow oil (4.35 g, 8.52 mmol, quantitative, not completely dry). UPLC-MS: t.sub.R=3.00 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=48.2%, ESI-MS: (calculated MH.sup.+: 511.24, MNa.sup.+: 534.23 g/mol, found: −).
3-(4-(2-bis-(tert-Butoxycarbonyl)-((aminooxy)acetyl)piperazin-1-yl)-4-nitrobenzyl(4nitrophenyl) carbonate (X13)
[0523] 2-bis-(tert-Butoxycarbonyl-aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrophenyl)piperazin-1-yl)ethan-1-one (2,06 g, 4 mmol) was provided in a 8 mL round bottom flask with a stirring bar. To the solution dry pyridine was added. Thereafter, 4-nitrophenylchloroformiate (0.86 g, 4.2 mmol) was added slowly as a solid at room temperature. The reaction is exothermic and may cause DCM to bubble. After 2 h stirring at room-temperature UPLC-MS and TLC indicated complete conversion to the carbonate. The solvent was removed under reduced pressure at a rotational evaporator. The product was purified via column chromatography (cyclohexane/ethylacetate 1:1) and gave a yellow solid (410 mg, 0.61 mmol). UPLC-MS: t.sub.R=3.44 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=92.4%, ESI-MS: (calculated MH.sup.+: 676.25 g/mol, MNa.sup.+: 698.23 g/mol, found: 698.51 m/z).
[0524] .sup.1H-NMR (400 MHz, CDCl.sub.3, 25° C.): δ [ppm]=8.29 (d, J=9.2 Hz, 2H), 7.84 (d, J=8.3 Hz, 1H), 7.39 (d, J=9.2 Hz, 2H), 7.19 (s, 1H), 7.16 (d, J=9.7 Hz, 1H), 5.29 (s, 2H), 4.61 (s, 2H), 3.89 (t, J=3.90, 2H), 3.78 (t, J=3.78, 2H), 3.15 (t, J=3.15, 2H), 3.10 (t, J=3.10, 2H), 1.54 (s, 18H).
Synthetic Steps for the Synthesis of 3-((4-(2-(aminooxy)acetyl)piperazin-1-yl)methyl)-4-nitrobenzyl (4-nitrophenyl) carbonate (X43)
[0525] 2-bis-(tert-Butoxycarbonyl)-(aminooxy)-1-(4-(5-(hydroxymethyl)-2-nitrobenzyl)piperazin-1-yl)ethan-1-one (1.27 g, not completely dry, assumed 1.86 mmol) was dissolved in 5 mL Chloroform in a 50 mL round bottom flask with a magnetic stirring bar and cooled to 0° C. with an ice bath. Dry pyridine (0.23 mL, 0.23 g, 2.88 mmol) and p-Nitrophenylchloroformate (0.59 g, 2.88 mmol) were added successively. After x h UPLC-UV/vis indicated complete conversion of the starting material. With a rotational evaporator the solvent was removed, and the residue was dissolved in 5 mL Ethyl acetate/Cyclohexane (1:1). After purification via flash column chromatography with Ethyl acetate/Cyclohxane (1:1) the desired product was obtained (0.02 g, 0.03 mmol). UPLC-MS: t.sub.R=2.96 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=55.6%, ESI-MS: (calculated MNa.sup.+: 712.24 g/mol, found: 712.41 m/z).
[0526] Chemical Synthesis of Amine Switch with Nucleophilic Release (Type 3) Linker Molecules X22 and X42
Synthesis of 2-(2-azido-5-(4-(2-((2-bis-(tert-Butoxycarbonyl)amino)oxy)acetyl)piperazin-1-yl)phenyl) acetic acid (X22)
Synthesis of methyl 2-(5-fluoro-2-nitrophenyl)acetate
[0527] 5-Fluoro-2-nitrophenylacetic acid (3.00 gm, 14.92 mmol) was taken in 100 ml round bottom flask with a stirring bar. The compound was dissolved in 40 ml of MeOH and 1.62 ml (29.84 mmol) of H.sub.2SO.sub.4 was added slowly at the room temperature. The resulting reaction mixture was refluxed for 6 h. The progress of the reaction was monitored by UPLC-MS and TLC. After completion of the reaction, Methanol was removed under reduced pressure at a rotational evaporator. To the resulting crude, 20 ml water was added, and the solution was neutralized by using saturated K.sub.2CO.sub.3 solution (50 ml). The precipitated solid was filtered and washed with water and dried under reduced pressure to obtain (129.86 mmol, 98%) desired product as a white color solid. UPLC-MS: t.sub.R=2.41 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=97%, ESI-MS: (calculated MH.sup.+: 214.05 g/mol, found: . . . ).
Synthesis of tert-butyl 4-(3-(2-methoxy-2-oxoethyl)-4-nitrophenyl)piperazine-1-carboxylate
[0528] Methyl 2-(5-fluoro-2-nitrophenyl)acetate (0.70 gm, 3.25 mmol) was dissolved in Dry DMF. To this solution, 1-Boc-piperazine (0.83 gm, 4.39 mmol) and Na.sub.2CO.sub.3 (0.71 gm, 6.5 mmol) was added at room temperature and the resulting reaction mixture was heated up to 80° C. for overnight. The reaction progress was monitored by UPLC-MS and TLC. After completion of reaction, the solution was filtered to remove the solid byproducts and the DMF was removed under reduced pressure using a rotational evaporator. Ice cold water (25 ml) was added to the crude material and resulting solid was filtered and washed with water (50 ml) and dried under reduced pressure to obtain expected product (1.01 gm, 2.66 mmol, 82%) in yellow color solid UPLC-MS: t.sub.R=3.01 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=96.0%, ESI-MS: (calculated MH.sup.+: 380.18 g/mol, found: 324.10(M2H.sup.+-tbu).
Synthesis of tert-butyl 4-(4-amino-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate
[0529] To a solution of the tert-butyl 4-(3-(2-methoxy-2-oxoethyl)-4-nitrophenyl)piperazine-1-carboxylate (1.00 gm, 2.58 mmol) in dioxane/H.sub.2O (25 mL, 3:1) was added NH.sub.4Cl (1.23 gm, 1.01 gm, 18.50 mmol) and Zn dust (1.23 gm, 18.50 mmol) at rt. The reaction mixture was stirred for 3 h at the same temperature, after that it was filtered through a celite bed. The resulting solution was evaporated, and crude material was partitioned between H.sub.2O (100 mL) and EtOAc (300 mL). The organic layer was separated, dried (MgSO.sub.4) concentrated to get desired product in Yellow color solid (1.75 mmol, 68%). UPLC-MS: t.sub.R=2.12 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=94.0%, ESI-MS: (calculated MH.sup.+: 350.21 g/mol, found: 350.19 m/z).
Synthesis of tert-butyl 4-(4-azido-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate
[0530] The tert-butyl 4-(4-amino-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (0.35 gm 0.99 mmol) dissolved in dry acetonitrile (25 mL) and cooled to 0° C. 90% tert-Butyl nitrite (0.837 gm, 7.94 mmol) was added dropwise to the reaction mixture and then TMSN.sub.3 (0.722 g, 5.95 mmol) was added over 10 minutes. The resulting red colored mixture was stirred for 3 h. The progress of the reaction was monitored by UPLC-MS. After completion of reaction, excess of TMSN.sub.3, t-BuONO and the solvent were removed under reduced pressure the obtained red residue was dissolved in 50 mL ethyl acetate and washed with (2×50 mL) water. The ethylacetae layer was dried over MgSO.sub.4 and removed under reduced pressure to get orange solid which was taken forward without further purification (0.33 gm, 0.88 mmol). UPLC-MS: t.sub.R=3.01 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=95.0%, ESI-MS: (calculated MH.sup.+: 376.20 g/mol, found: 376.28 m/z).
Synthesis of 2-(2-azido-5-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)acetic acid
[0531] A solution of the tert-butyl 4-(4-azido-3-(2-methoxy-2-oxoethyl)phenyl)piperazine-1-carboxylate (0.314 gm, 0.83 mmol), LiOH (0.102 gm, 4.14 mmol), MeOH (5 mL), and H.sub.2O (0.2 mL) was stirred at rt for 3 h. After complete conversion of the starting materials to the product (monitored by UPLC-MS), the reaction mixture was evaporated and to the resulting crude material, saturated NH.sub.4Cl added till the solution PH reaches to 6. The resulting solution was extracted with EtOAc (2×25 mL), dried over MgSO.sub.4 and concentrated to provide the product as a Yellow solid (0.65 mmol, 78%). UPLC-MS: t.sub.R=2.55min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=95.0%, ESI-MS: (calculated MH.sup.+: 362.18 g/mol, found: 306.04 m/z).
Synthesis of 4-(4-azido-3-(carboxymethyl)phenyl)piperazin-1-ium 2,2,2-trifluoroacetate
[0532] Pure trifluoracetic acid (0.6 ml, 6.14 mmol) was added dropwise to the round bottom flask containing 2-(2-azido-5-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)acetic acid (0.22 gm, 0.61 mmol) and the resulting solution was stirred at rt for 1 hour. After completion of reaction (monitored by UPLC-MS), cold ether (25 mL) was added and the resulting solid was filtered and washed with cold ether. The resulting light brown color solid (0.30 gm, 85%) was dried and proceeded to further steps without purification. UPLC-MS: t.sub.R=2.80 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=87.0%, ESI-MS: (calculated MH.sup.+: 262.13 g/mol, found: 262.11 m/z) wrong mass.
Synthesis of 2-(2-azido-5-(4-(2-((((2-bis-(tert-Butoxycarbonyl)amino)oxy)acetyl)piperazin-1-yl)phenyl) acetic acid (X22)
[0533] Bis-(tert-Butoxycarbonyl)-(aminooxy)acetic acid ((Boc).sub.2AOAcOH, 88.4 mg, 0.30 mmol) and N-hydroxysuccinimide (NHS, 34.6 g, 0.30 mmol) was dissolved in 2 mL dry Acetonitrile. To this solution was added dicyclohexylcarbodiimide (DCC, 62.0 mg, 0.30 mmol) as a solid, after dissolution of DCC a white precipitate formed. The reaction mixture was stirred for 1 h at room temperature where the (Boc).sub.2AOAc-NHS ester was quantitatively formed according to UPLC-MS. Thereafter the mixture was filtered with a filter paper to remove the DCC-urea directly into a reaction flask that contains 4-(4-azido-3-(carboxymethyl)phenyl)piperazin-1-ium 2,2,2-trifluoroacetate (100 mg, 0.20 mmol) in dry DMF (2 ml) and DIEPA (155 μL, 0.89 mmol). The mixture was further stirred at room temperature for 1-2 h. After completion of the reaction, Solvent was evaporated under reduced pressure and reaction mixture was neutralized by adding NH.sub.4Cl (25 ml). The compound was extracted with EtOAc (2×25 mL), dried over MgSO.sub.4 and concentrated to provide the product as a dark brown color solid (0.19 mmol, 97%). UPLC-MS: t.sub.R=2.80 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=87.0%, ESI-MS: (calculated MNa.sup.+: 557.23 g/mol, found: 557.28 m/z).
[0534] .sup.1H NMR (400 MHz,) δ 7.06 (d, J=8.7 Hz, 1H), 6.90 (d, J=8.8 Hz, 1H), 6.83 (s, 1H), 4.60 (s, 2H), 3.90-3.84 (m, 2H), 3.77-3.72 (m, 2H), 3.59 (s, 2H), 3.24-3.11 (m, 4H), 1.54 (s, 18H).
Synthesis of 2-(5-(4-(2-((2-bis-)tert-Butoxycarbonyl)amino)oxy)acetyl)piperazin-1-yl)-2-nitrophenyl)acetic acid (X42)
Synthesis of 2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-nitrophenyl)acetic acid
[0535] To the solution tert-butyl 4-(3-(2-methoxy-2-oxoethyl)-4-nitrophenyl)piperazine-1-carboxylate (0.50 gm, 1.30 mmol) in MeOH: H.sub.2O(21.2 mL, 16:1), LiOH was added and stirred at rt for overnight. After complete conversion of the starting materials to the product (monitored by UPLC-MS), the reaction mixture was evaporated and to the resulting crude material saturated NH.sub.4Cl (25 mL) added till the solution PH reaches to 6. The compound was extracted with EtOAc (4×25 mL), dried over MgSO.sub.4 and concentrated under reduced pressure to provide the desired product as a Yellow solid (0.78 mmol, 59%). UPLC-MS: t.sub.R=2.36 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=97.0%, ESI-MS: (calculated MH.sup.+: 380.18 g/mol, found: 310.04 m/z (2H.sup.+-tbu m/z)).
Synthesis of 4-(3-(carboxymethyl)-4-nitrophenyl)piperazin-1-ium 2,2,2-trifluoroacetate
[0536] Pure trifluoracetic acid (0.73 ml, 7.59 mmol) was added dropwise at the room temperature to the round bottom flask containing 2-(5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-nitrophenyl)acetic acid (0.28 gm, 0.76 mmol) and the resulting solution was stirred for 1 hour. After completion of reaction (monitored by UPLC-MS), cold ether (25 mL) was added and the resulting solid was filtered and washed with cold ether. The resulting Yellow color solid (88%) was dried and proceeded to further steps without purification. UPLC-MS: t.sub.R=1.35 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=88.9%, ESI-MS: (calculated MH.sup.+: 266.11 g/mol, found: 266.11 m/z).
Synthesis of 2-(5-(4-(2-((2-bis-)tert-Butoxycarbonyl)amino)oxy)acetyl)piperazin-1-yl)-2-nitrophenyl)acetic acid (X42)
[0537] Bis-(tert-Butoxycarbonyl)-(aminooxy)acetic acid ((Boc).sub.2AOAcOH, 88.4 mg, 0.30 mmol) and N-hydroxysuccinimide (NHS, 34.6 g, 0.30 mmol) was dissolved in 2 mL dry Acetonitrile. To this solution was added dicyclohexylcarbodiimide (DCC, 62.0 mg, 0.30 mmol) as a solid, after dissolution of DCC a white precipitate formed. The reaction mixture was stirred for 1 h at room temperature where the (Boc).sub.2AOAc-NHS ester was quantitatively formed according to UPLC-MS. Thereafter the mixture was filtered with a filter paper to remove the DCC-urea directly into a reaction flask that contained 4-(3-(carboxymethyl)-4-nitrophenyl)piperazin-1-ium 2,2,2-trifluoroacetate (100mg, 0.20 mmol) in dry DMF (2 ml) in and DIEPA (155 μL, 0.89 mmol). The mixture was stirred at room temperature for 1-2 h after which the completion of the reaction was confirmed by UPLC-MS. After completion of the reaction, Solvent was evoparated under reduced pressure and reaction mixture was neutralized by adding NH.sub.4Cl (25 ml). The resulting solution was extracted with EtOAc (2×25 mL) and concentrated to provide the product as a dark brown color solid (0.19 mmol, 97%). UPLC-MS: t.sub.R=2.74 min (10-90% MeCN in 3 min), UPLC-purity (278 nm)=87.0%, ESI-MS: (calculated MNa.sup.+: 561.22 g/mol, found: 561.28 m/z).